Carrier‐Free Self‐Assembly Nano‐Sonosensitizers for Sonodynamic‐Amplified Cuproptosis‐Ferroptosis in Glioblastoma Therapy

Abstract Cuproptosis is a newly discovered form of programmed cell death significantly depending on the transport efficacy of copper (Cu) ionophores. However, existing Cu ionophores, primarily small molecules with a short blood half‐life, face challenges in transporting enough amounts of Cu ions into tumor cells. This work describes the construction of carrier‐free nanoparticles (Ce6@Cu NPs), which self‐assembled by the coordination of Cu2+ with the sonosensitizer chlorin e6 (Ce6), facilitating sonodynamic‐triggered combination of cuproptosis and ferroptosis. Ce6@Cu NPs internalized by U87MG cells induce a sonodynamic effect and glutathione (GSH) depletion capability, promoting lipid peroxidation and eventually inducing ferroptosis. Furthermore, Cu+ concentration in tumor cells significantly increases as Cu2+ reacts with reductive GSH, resulting in the downregulation of ferredoxin‐1 and lipoyl synthase. This induces the oligomerization of lipoylated dihydrolipoamide S‐acetyltransferase, causing proteotoxic stress and irreversible cuproptosis. Ce6@Cu NPs possess a satisfactory ability to penetrate the blood‐brain barrier, resulting in significant accumulation in orthotopic U87MG‐Luc glioblastoma. The sonodynamic‐triggered combination of ferroptosis and cuproptosis in the tumor by Ce6@Cu NPs is evidenced both in vitro and in vivo with minimal side effects. This work represents a promising tumor therapeutic strategy combining ferroptosis and cuproptosis, potentially inspiring further research in developing logical and effective cancer therapies based on cuproptosis.


Instruments
Powder X-ray diffraction patterns of samples were recorded on a Rigaku Miniflex-600.
Fluorescence imaging by using confocal laser scanning microscopy (CLSM, Zeiss 800).The UV absorbance of samples were measured by ultraviolet visible (UV-Vis) spectrophotometer (Agilent).Metal content analysis by using inductively coupled plasma mass spectrometer (ICP-MS, PlasmaQuad 3, Thermo Elemental).Dynamic light scattering (DLS) and Zeta potential were obtained by using a Brook haven.The cell death was analyzed by flow cytometer (CytoFLEX, Beckman).
Synthesis of Ce6@Cu nanoparticles (Ce6@Cu NPs) The Ce6@Cu NPs were fabricated via a self-assembled strategy.10 mg Ce6 was dissolved in dimethylsulfoxide (DMSO), and then 4 mL 10 mg/mL CuCl2 were dropwise added into the Ce6 solution under mild stirring.After stirring for 24 h, the as-prepared Ce6@Cu NPs were dialyzed for 24 h.
To further confirm the Ce6@Cu NPs-catalyzed 1 O2 generation, electron spin resonance (ESR) analysis was employed using TEMP as the spin trapper.40 μg/mL Ce6@Cu NPs, and 10 mM TEMP was irradiated using US (1 W/cm 2 ) for 5 min.Then, the mixture was transferred to a quartz tube for ESR measurement.

GSH depletion capability of Ce6@Cu NPs
The GSH depletion capacity of Ce6@Cu NPs was evaluated using a probe DTNB. 1 mM GSH, 100 μg/mL Ce6@Cu NPs, and 1 mM DTNB incubated for 60 min.Then the absorption spectra were analyzed.

Cellular uptake
U87MG cells were seeded in confocal dishes for 12 h.After 4 h of incubation with Ce6@Cu NPs, tumor cells were co-stained with 10 μM Hoechst and 10 μM Lyso-tracker for 20 min.The fluorescence imaging of U87MG cells was imaged by CLSM.

Cytotoxicity assessments
Cell-viability was measured by the CCK-8 assay, live/dead cell staining assay, and flow cytometry analysis.For CCK-8 assay, U87MG cells were planted for 24 h.Then, the cells were incubated with various concentrations of Ce6 and Ce6@Cu NPs without/with US irradiation.After treatment for 24 h, the medium was replaced with fresh medium containing 10 μL CCK-8 and quantified by the absorbance at 450 nm using a microplate reader.
Live/dead cell staining assay was monitored by CLSM to observe the toxicity.U87MG cells were seeded in confocal dishes and incubated for 12 h.After 24 h of exposure to Ce6@Cu NPs without/with US irradiation (1 W/cm 2 ), the cells were co-stained with calcein-AM and PI for 20 minutes.The fluorescence imaging of cells was observed by confocal microscopy.
For analysis of cell death, Annexin V-FITC and PI kit was carried out.U87MG cells were seeded and incubated 12 h.Subsequently, the cells were exposed to Ce6@Cu NPs without/with US irradiation.
After co-staining with Annexin V-FITC and PI according to the manufacturer's protocols.The quantitative cell death was determined by flow cytometry.

In vitro reactive oxygen species (ROS) generation
U87MG cells were seeded in confocal dish.After incubation for 12 h, the cells were treated with different formulations for 4 h.Then, the cells were co-stained with DCFH-DA (10 μM) and Hoechst (10 μM).After 20 minutes of incubation, the medium was removed and the cells were washed with DMEM.
The fluorescence imaging of cells was imaged by confocal microscopy.

Intracellular GSH and GSSG content
U87MG cells were plated in 6-well plates and incubated for 24 h.Subsequently, the cells were exposed to Ce6@Cu NPs without/with US irradiation.The GSH and GSSG contents were measured using a DTNB kit.The assay was carried out according to the manufacturer's instructions.The absorbance of 340 nm was measured by a microplate reader.

Change of mitochondrial membrane potential (MMP)
To investigate the MMP, U87MG cells were seeded and incubated for 24 h.Subsequently, the cells were exposed to Ce6@Cu NPs without/with US irradiation.Then the cells were treated according to the JC-1 kit.The fluorescence imaging of cells was analyzed by confocal microscopy.

Lipid peroxidation (LPO) initiated by Ce6@Cu NPs
The cellular LPO assay was carried out by using a BODIPY 581/591 -C11 probe.U87MG cells were seeded and incubated for 24 h.Subsequently, the cells were exposed to Ce6@Cu NPs without/with US irradiation.Then the cells were stained with BODIPY 581/591 -C11 probe and Hoechst for 30 min.The fluorescence imaging of cells was imaged by CLSM.

Figure S4 .
Figure S4.The stability of the Ce6@Cu NPs in DMEM and fetal bovine serum (FBS).

Figure S5 .
Figure S5.Drug release curve of Ce6@Cu NPs in different media.

Figure S10 .
Figure S10.Fluorescence images of U87MG cells incubated with Ce6 at different time points.

Figure S11 .
Figure S11.Bar graph showing the Cu content in U87MG cells incubated with Ce6@Cu NPs.

Figure S12 .Figure S13 .
Figure S12.CLSM images of colocalization in U87MG cells between the lysosome tracker (green channel) and Ce6@Cu NPs (red channel) after 4 h of incubation.

Figure S14 .
Figure S14.CLSM images of AO-stained U87MG cells after 24 h of incubation with various formulations.

Figure S15 .
Figure S15.CLSM images of GPX4 expression in U87MG cells incubated with various formulations for 24 h.

Figure S16 .
Figure S16.CLSM images of the oligomerization of lipoylated DLAT cultured with various formulations for 24 h.

Figure S17 .
Figure S17.CLSM images depicting FDX1 expression in U87MG cells following a 24-hour incubation with various formulations.

Figure S18 .
Figure S18.CLSM images depicting LIAS expression in U87MG cells following a 24-hour incubation with various formulations.

Figure S20 .
Figure S20.Haematoxylin and eosin (H&E)-stained images of major organs harvested from different groups of mice at 12 days post-treatment.

Figure S22 .
Figure S22.The corresponding quantification fluorescence intensity of the major organs (heart, liver, spleen, lung, kidney, and brain).

Figure S23 .
Figure S23.Kaplan-Meier survival curves for various groups of mice.

Figure S24 .
Figure S24.The body weights of mice bearing tumors during the treatment period.

Figure S25 .Figure S26 .
Figure S25.H&E staining was performed on brains collected from various groups at different time points.

Figure S27 .
Figure S27.Ki67 staining of brain tumors collected from different groups.

Figure S28 .
Figure S28.Immunochemical staining of brain tumors for LIAS expressions in different groups.

Figure S29 .
Figure S29.Immunofluorescent staining of brain tumors to assess the expression levels of GPX4 in different groups.